A total of 46 consecutive patients with primary EC were enrolled in this study. They consisted of 38 males (82.6%) and 8 females (17.4%), with a mean age of 56.4 years (range, 46-70 years), who were treated at Gansu Provincial Tumor Hospital between April 2005 and June 2007. The patients’ eligibility for this study was as follows: (1) Primary lesion was squamous carcinoma in histology. There was no previous malignant disease or clinical evidence of a second primary tumor elsewhere; (2) Karnofsky performance status ≥ 70, white blood cell and hemoglobin levels within the normal range; and (3) Having not received radiotherapy, chemotherapy and operation. Tumor, nodes and metastasis (TNM) classification was established on the basis of pathologic examinations of biopsied specimens. Lymph node status in the patients was determined by computed tomography (CT). According to the UICC (2002) classification, 10 patients were in stage I, 17 in stage II, 14 in stage III and 5 in stage IV. Twenty-eight patients had no signs of lymph node metastasis, and the other 18 patients had metastasis of mediastinal and/or supraclavicular lymph nodes. Thirty-two had primary lesions < 6 cm and 14 were ≥ 6 cm in size. Five tumors were located in the upper thorax, 21 in the middle thorax and 20 in the lower thorax. To compare the reliability of seronegative testing, a total of 30 healthy volunteers, 19 males and 11 females, with a mean age of 36 years (range, 21-62 years) from our hospital staff and medical students served as controls. Informed consent was obtained from all patients. The blood samples were collected on the day before radiotherapy and on the administration of an irradiation dose of 20 Gy/10 f/12 d, 40 Gy/20 f/24 d and 60 Gy/30 f/36 after radiotherapy, and stored at -80°C until Assay.

Radiation source was a 6MV linear accelerator. The design of the radiation fields was based on the diagnosis by CT and barium examination. For all patients, a three-field approach was administered: one anterior and two posterior oblique portals. The width of the fields was adjusted to cover gross tumors with 3-4 cm extended margins so as to include subclinical lesions. The length of the field covered clinical tumors with a 3-5 cm extended margin at both ends of the lesion. All patients received conventional fractions, 2.0 Gy per fraction, five fractions per week. The total dose given to the tumor was 60-70 Gy/6-7 W.

Anti-p53 antibodies in sera of patients was detected using a commercially available sandwich enzyme-linked immunosorbent assay kit (Immunotech, France). The assay was done according to the manufacturer’s instructions, with the following specifications: patient serum was added for 60 min at 37°C to microtiter wells coated with recombinant wild type human p53 protein to detect specific anti-p53 antibodies or with a control protein to detect nonspecific interactions. After washing, goat antihuman immunoglobulin G (IgG) antibody conjugated with peroxidase was added and stayed for 60 min at 37°C. Next, the substrate 3,3,5,5-tetramethylbenzidine (TMB) was added for 10 min. The enzymatic process was halted by adding 2N hydrogen chloride. Light absorption was measured at 450 nm using a photospectrometer. Because human serum may give rise to variable background signals, an internal control was used to measure the nonspecific background of each sample. This background reflects nonspecific interactions of serum components either with the plastic or with the components used in the ELISA. In the assay, the nonspecific background corresponds to the absorbance measured on wells coated with control protein. The presence of anti-p53 antibodies in a sample is determined by different parameters, i.e., for the specific signal of the sample, index = specific signal of the positive control (low); for p53 net absorbance of the sample, ratio of net absorbance = control protein net absorbance of the sample. An index of 1.1 and a ratio of net absorbance of 1.6 were determined to be positive for the presence of anti-p53 antibodies.

At the end of RT, all patients received esophageal barium examination and the clinical radiation response was evaluated according to standard X-ray diagnosis of EC after radiotherapy[12]. A complete response (CR) was defined as the disappearance of the mass shadow, no narrowing observed in the esophageal lumen, and none or slight rigidity of the esophageal wall without residual ulceration. Partial response (PR) was > 50% reduction in tumor bulk, but < 100% resolution of the disease and a residual shallow ulcer with a diameter < 1.5 cm, despite the disappearance of the mass shadow. No response (NR) was defined as no improvement in the X-ray findings, with a deep and large residual ulcer or complete obstruction of the esophageal lumen, regardless of the residual state of the mass shadow.

The Statistical Package for Social Sciences, version 10.0 was used for statistical analysis. Serum p53-Abs levels were expressed as means ± SD. Student’s t test, Pearson Chi-square and logistic analysis were used to determine the significance of difference between the two groups. A P value < 0.05 was considered significant.

The index and ratio of serum p53-Abs for EC patients were 1.5847 ± 0.5133 and 3.0293 ± 0.7013, and those for healthy controls were 0.2418 ± 0.1438 and 1.0361 ± 0.2175, the former was higher than the latter (P < 0.0001).

The positive rate of serum p53-Ab was 39.1% (18 of 46) for EC patients and 0% (0 of 30) for healthy controls, with a significant difference (P < 0.0001). The sensitivity, specificity, accurate rate, positive predictive value and negative predictive value of p53-Ab detection in EC were 39.1%, 100%, 63.2%, 100% and 52%, respectively.

The level and positive rate of serum p53-Abs had significant differences between before radiotherapy, after administration of an irradiation dose of 40 Gy/20 f/24 d and after administration of an irradiation dose of 60 Gy/30 f/36 d (Table 1).

The positive rate of serum p53-Abs in EC was related to histological grade, disease stage and lymph node metastasis, but it was not significantly related to sex, age and to the size and site of tumor (Table 2).

Logistic analysis showed that the positive rate of serum p53-Abs in EC had positive correlation with disease stage, negative correlation with histological grade and independence with sex, age and with the size and site of tumor (Table 3).

At the end of RT, CR was achieved in 21 patients, RR in 18 and NR in 7. In patients with CR plus PR, 61.1% (11/39) were positive for serum p53-Abs, but 100% (7/7) of patients with NR were positive for serum p53-Abs. The positive rate of serum p53-Abs in EC patients with effect was significantly lower than those without effect after radiotherapy (P < 0.0001).

Mutations of the p53 gene are common gene alterations in most malignant tumors, including esophageal carcinoma. Mutant p53 protein is accumulated in the cell because of its longer half-life compared with wild-type protein. p53 protein plays a crucial role in the regulation of the cell cycle and has been implicated in cell differentiation, apoptosis, DNA synthesis and repairment. In addition, it has been shown that patients with various types of neoplasias have p53 antibodies in their sera. ELISA was used to detect anti-p53 antibodies in the sera. The authors explored the relationship between serum p53-Abs and clinicopathological characteristics and therapeutic effect in patients with esophageal carcinoma, and investigated the sequential changing regularity of serum p53-Abs after radiotherapy.

Studies have revealed that overexpression of p53 protein is closely related to induction of drug resistance in cancer patients and it can be used as a predictor for chemosensitivity to tumor treatment. Specific p53-Ab was found in the sera of cancer patients with p53 protein overexpression. Furthermore, the presence of serum p53-Ab is closely correlated with p53 protein overexpression. Thus, serum p53-Ab could theoretically be useful in predicting radiosensitivity in cancer patients. However, the clinical relevance of p53-Ab and radiotherapy for esophageal carcinoma has not been fully studied.

From the results of this study, the following conclusions can be drawn that the positive rate of serum p53-Abs in esophageal carcinoma is related to histological grade, disease stage and lymph node metastasis, but it is not significantly related to sex, age and the size and site of tumor. The level and positive rate of serum p53-Abs had significant differences between before radiotherapy, after administration of an irradiation dose of 40 Gy/20 f/24 d and after administration of an irradiation dose of 60 Gy/30 f/36 d. The positive rate of serum p53-Abs in esophageal carcinoma patients with effect is significantly lower than those without effect after radiotherapy.

Detection of serum p53-Abs is helpful to diagnosis of esophageal carcinoma. Monitoring of sequential change of serum p53-Abs before and after radiotherapy in patients with esophageal carcinoma is also useful in evaluating the response to the treatment and prognosis of the patients.

The authors detected the serum p53 antibody level in patients with esophageal squamous cell carcinoma and its sequential changes before and after radiotherapy. The results showed that changes of serum p53-Abs are helpful to the diagnosis of esophageal carcinoma. Monitoring of sequential change of serum p53-Abs before and after radiotherapy in patients with esophageal carcinoma is helpful to evaluating the response to the treatment and prognosis of the patients. Therefore, this study has values in clinical management of the patients.